Investigating short-term toxicity of melphalan in a model of an isolated and superfused bovine retina.

BACKGROUND: Melphalan, as a treatment for retinoblastoma, has been applied intra-arterially by catheterisation of the ophthalmic artery or intravitreally, aiming to reduce systemic side effects of intravenous drug therapy. This study evaluates retinal toxicity of different melphalan concentrations measured by electroretinogram (ERG) in an isolated and perfused retinal whole mount culture. METHODS: For functional testing, bovine retinas were prepared and perfused with an oxygen-saturated standard solution and the ERG was recorded until stable b-wave or a-wave amplitudes were reached. Thereafter, retinae were exposed to 80, 160 and 320 µg/ml of melphalan for 30 min. After exposure, a washout was performed thrice for 5 min each and the ERG amplitude recovery was monitored for 60 min. To investigate the effects on photoreceptor function, 1-mM asparate was added to suppress the b-wave and obtain isolated a-waves. RESULTS: While no toxic effects for a concentration of 80 µg/ml were observed, both b- and a-waves were significantly reduced after application of 160 (b-wave 43.8 %, p = 0.03; a-wave 28.2 %, p = 0.04) and 320 µg/ml (b-wave 20.0 %, p = 0.04; a-wave 35.8 %, p = 0.02). For 320 µg/ml, this reduction remained significant at the end of the washout (b-wave 40.0 % p = 0.02; a-wave 26.4 %, p = 0.02). CONCLUSIONS: Epiretinal or intraretinal concentrations of 80-µg/ml melphalan do not cause toxic effects in this in vitro model. Concentrations higher than 160 µg/ml should be avoided.

Toxic effects of melphalan, topotecan and carboplatin on retinal pigment epithelial cells.

PURPOSE: Clinical evidence of retinal pigment epithelium (RPE) alterations after intra-arterial (IAC) and intravitreal chemotherapy (IViC) of retinoblastoma has been reported. We, therefore, investigated the cellular toxic effects of melphalan, topotecan and carboplatin on the RPE in a cell culture model. METHODS: The effects of melphalan, carboplatin and topotecan on ARPE19 cell morphology were examined by phase contrast microscopy. Cell proliferation was quantified by BrdU incorporation, cell viability studied via MTS assays, and cell densities were estimated by Crystal Violet staining, and apoptosis induction studied via caspase 3/7-activity assays after a 24-hr incubation period. Staurosporine, media without fetal bovine serum, diluents of melphalan, carboplatin and topotecan were applied as positive and negative controls, respectively. RESULTS: We observed a concentration-dependent increase in the number and size of gaps in the ARPE19 cell layer with each drug. There was a significant decrease in proliferative activity and cell viability of RPE cells as well as an increase in apoptosis after 24 hrs culture in media supplemented with melphalan and topotecan. Carboplatin had comparable effects on cell proliferation and cell viability; however, no significant apoptotic impacts were observed. The three cytostatic drugs had insignificant effects on cell density measurements. CONCLUSIONS: Morphological monitoring and toxicity assays indicate a direct toxic effect of melphalan and the other two cytostatic drugs on ARPE19 cells. Thus, a direct toxic effect of melphalan in vivo after IAC or IViC on the RPE seems probable and may explain the clinical and angiographic RPE alterations observed in some retinoblastoma patients.

Primary vitrectomy for rhegmatogenous retinal detachment in pseudophakic eyes: 20-gauge versus 25-gauge vitrectomy.

PURPOSE: To report anatomical and functional outcome of 20-gauge versus 25-gauge primary pars plana vitrectomy for management of complex rhegmatogenous retinal detachment in pseudophakic eyes. METHODS: Prospective single-centre randomized comparative pilot trial. Fifty patients with retinal detachment (RD) not complicated by proliferative vitreoretinopathy grade B or C, who cannot be treated with a single meridional sponge, were randomized (1:1) from November 2006 to January 2010 to either 20-gauge or 25-gauge vitrectomy as first surgical intervention and followed up over a 12-month period, evaluating change in best-corrected visual acuity, anatomical success and intraocular pressure dysregulation. RESULTS: Mean visual acuity improved by 0.88 (SD 0.67) from 1.22 logMAR (SD 0.63) to 0.34 logMAR (SD 0.31) in the 20-gauge group and by 0.53 (SD 0.91) from 0.86 logMAR (SD 0.73) to 0.34 logMAR (SD 0.46) in the 25-gauge group. Final anatomical success rate was 100% and primary success rate was 69% at 6 months of follow-up. In the 20-gauge group, the retina was attached after one single procedure in 18 eyes (72%) and in 21 eyes (84%) of the 25-gauge group. Two patients in the 25-gauge group had hypotony at the first postoperative day which normalized within 6 weeks. CONCLUSION: In our series, transconjunctival sutureless 25-gauge and 20-gauge vitrectomy showed comparable results in pseudophakic RD not suitable for single sponge surgery with respect to visual outcome and retinal reattachment. Postoperative hypotony does not seem to be a significant problem of transconjunctival sutureless vitrectomy.

Electrophysiological toxicity testing of VEGF Trap-Eye in an isolated perfused vertebrate retina organ culture model.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of visual impairment in Western nations. Since the discovery of the importance of vascular endothelial growth factor (VEGF) in the pathogenesis of neovascular AMD, anti-VEGF agents including pegaptanib, ranibizumab and bevacizumab provide a treatment option to improve vision in affected persons. VEGF Trap-Eye (Aflibercept) is a new agent available for the treatment of exudative AMD. The molecule is a receptor decoy with a longer half-life and a higher affinity to VEGF compared with ranibizumab or bevacizumab. The presented study has been designed to evaluate the short-term toxic effects of VEGF Trap-Eye on retinal function during and after direct exposure to the drug. METHODS: Isolated bovine retinas were perfused with an oxygen-saturated nutrient solution, and the electroretinogram (ERG) was recorded using silver/silver chloride electrodes. A total of 0.5 mg or 2 mg VEGF Trap-Eye was added to the nutrient solution and retinas were exposed for 45 min, followed by a washout period of 100 min. The percentage of a- and b-wave reduction at the end of the washout was compared with the baseline values. Additionally, retinal whole mount cultures were exposed for 24 hr to VEGF Trap-Eye, and the amount of apoptotic cells were determined using the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labelling (TUNEL) assay. RESULTS: During simulation of intraocular application, no significant reduction in the a-wave amplitude for 0.5 mg (2.70%, p = 0.37) and 2 mg (3.84%, p = 0.37) VEGF Trap-Eye and b-wave amplitude for 0.5 mg (19.68%, p = 0.17) and 2 mg (24.1%, p = 0.06) VEGF Trap-Eye was observed at the end of the washout. However, there were significant changes in a-wave and b-wave amplitudes directly after exposure to 0.5 mg VEGF Trap-Eye (18.4%, p = 0.004 and 43.1%, p = 0.006, respectively). CONCLUSIONS: The presented data suggest that intraocular application of up to 2 mg VEGF Trap-Eye does not induce irreversible toxic retinal damage. However, short-term results showed a negative effect directly after the application for 0.5 mg and 2 mg VEGF Trap-Eye.

Comparative toxicity and proliferation testing of aflibercept, bevacizumab and ranibizumab on different ocular cells.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is a key factor in the pathogenesis of neovascular retinal diseases including age-related macular degeneration. VEGF inhibitors including ranibizumab, pegaptanib or bevacizumab improve retinal morphology and vision in many patients. The recently approved drug aflibercept (VEGF Trap-Eye/Eyelea, Regeneron, Tarrytown, New York, USA) offers a new therapy modality. We therefore tested for toxic and anti-proliferating effects of aflibercept. METHODS: The effects of aflibercept (0.125, 0.5, 2 mg), ranibizumab (0.125 mg) and bevacizumab (0.3125 mg) after 1, 24, 48 and 72 h on cell morphology via phase contrast pictures, cell viability via MTS assay, total cell amount via crystal violet staining, apoptosis induction via caspase 3/7 assay and proliferation via BrdU assay were investigated. Three ocular cell lines were chosen for toxicology testing: ARPE19 cells, RGC-5 cells and 661W cells. RESULTS: Aflibercept did not cause changes in cell morphology, induce apoptosis or cause permanent decrease in cell viability, cell density or proliferation in any cell line or concentration investigated. In general, aflibercept had fewer effects (upregulation or downregulation) compared with controls than bevacizumab or ranibizumab. CONCLUSIONS: In our experiments, aflibercept did not lead to any negative effects on retinal cell lines and might therefore be used safely in clinical applications.